Use of psychrophilic anaerobic digestion in sequencing batch reactor for degradation of prions

ABSTRACT

The present invention relates to the use or process of use of a sequencing batch reactor for eliminating prion in specified risk materials and for measuring the efficacy of a sequencing batch reactor to degrade prion proteins in specified risk materials.

TECHNICAL FIELD

The present invention relates to the use or process of use of asequencing batch reactor for eliminating prion in specified riskmaterials.

BACKGROUND ART

Prions are proteins devoid of nucleic acids and cell membranes which arelargely unaffected by standard methods of sterilisation. Prions areunprecedented infectious pathogens that cause a group of invariablyfatal neurodegenerative diseases by an entirely novel mechanism(Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383). Inactivation ofprions poses significant environmental and health issues both for thedisposal of prions infected animals and in the preparation of materialsof animal origin, such as animal feed. Slaughterhouse sludge and animalmortalities (carcasses) are an important source of pathogens orinfectious prions proteins.

Prions are protein naturally found in animals. Cellular or normal formof prion proteins are constitutively expressed in the brains of healthyadult animals, but are highly regulated, both spatially and temporally,during development (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383).There are two isoforms of prion proteins. PrPC (Prion Protein Cellularisoform) is a cell surface, N-linked, α-helices-rich, globular solubleglycoprotein protein that may serve as a signal transduction protein(Mouillet-Richard et al., 2000, Science, 289: 1925-1928) and may play anessential role in the normal development of mammal brain. PrPC occursboth in healthy and diseased tissues. PrPSc (Prion Protein Scrapieagent) is a β-sheet-rich, fibrous, highly insoluble protein which is thecausative agent of central nervous system diseases in mammals. It isthought that a higher content in β-sheet confer heat- andprotease-resistance to PrPSc.

The presence of the abnormal, pathological isoform (PrPSc) is typical ofthe diseased state. PrPC and PrPSc are identical in their primarystructure. Differences occur in secondary structures: PrPC containsthree α-helices (about 40% α-helix) and a short antiparallel β-sheet,whereas PrPSc is composed of two α-helices (about 30% α-helix) and fourβ-sheets (45% β-sheets) (Prusiner, 1998, Proc Natl Aca Sci, 95:13363-13383)). This induces conformational changes in the tertiarystructure of PrPSc, resulting in modified characteristics. Both PrPisoforms are devoid of nucleic acids (Prusiner, 1998, Proc Natl Aca Sci,95: 13363-13383).

Experimental data suggests that prions can survive for a long time innatural environments. For example, Brown and Gajdusek (1991, J InfectDis, 153: 1145-1148) showed that prions buried into a garden soil couldsurvive and retain their infectivity power for 3 years, with littleleaching deeper into the soil.

Current research suggests that the primary method of infection inanimals is through ingestion. It is thought that prions may be depositedin the environment through the remains of dead animals and via urine,saliva, and other body fluids. They may then linger in the soil bybinding to clay and other minerals. Sterilizing prions involves thedenaturation of the protein to a state where the molecule is no longerable to induce the abnormal folding of normal proteins. However, prionsare generally quite resistant to proteases, heat, radiation, andformalin treatments, although their infectivity can be reduced by suchtreatments. Effective prion decontamination relies upon proteinhydrolysis or reduction and/or destruction of protein tertiarystructure. Examples include bleach, caustic soda, or strong acidicdetergents.

Transmissible Degenerative Encephalopathies. (TDE) forms a group offatal neurodegenerative disorders caused by the accumulation of prionsin the brains of mammals. TDE are unique in that the host's normal prionprotein (PrPc) is modified into the infective prion protein (PrPsc) as aconsequence of infection (Carp et al., 1985, J Gen Virol, 66:1357-1368), and forms deposits in affected tissues, especially in thecentral nervous system. TDE affect a wide variety of wild animals andlivestock, as well as humans, and present in 3 ways, all of whichinvolve modifications of the prion protein: heritably as a result ofgenetic mutations; sporadically by spontaneous conversion of the prionprotein into a pathologic form via yet undefined mechanisms; byinfection following exposure to the exogenous misfolded form of theprion protein.

Bovine Spongiform Encephalopathy (BSE) is among the most notable priondisease. The International Trade Commission (ITC) released a reportestimating that trade restrictions resulting from Bovine BSE cost thecattle industry $11 billion from 2004 to 2007.

Thus, not only are animal manure management practices are oftendetrimental to the environment, they also represent a potential hazardto human and animal health, in addition in producing strong odours,encourage fly breeding, induce weed problems and pollute air, soil andwater.

The Canadian Food Inspection Agency estimated the amount of specifiedrisk materials (SRM) generated in Canada at 170,000 tonnes annually.Safe disposal of SRM potentially contaminated with prions is challengingsince these TDE-causing agents are relatively resistant to inactivationby physical or chemical procedures usually applied for microorganisms(Taylor et al., 1994, Arch Virol, 139: 313-326). Moreover, significantcosts are associated with some disposal treatment. Physical and chemicalmethods of prions inactivation that efficiently and/or rapidly fixproteins, including alcohols, aldehydes and rapid heating with steam,protect prions from inactivation, hence enhancing their thermostability. Thus, treatments that disrupt protein structure, rather thanfixing it, are required when considering the disposal of SRM potentiallycontaminated with prions. Moreover, treatments using chemicals such asdenaturants, detergents, strong alkali are inappropriate for theinactivation of prions in SRM due to potential user exposure at the farmand disposal problems onto agricultural land.

Consequently, there is a need to develop biological treatments for thedegradation of prions in SRM which are environmentally sound.

It would be thus highly desirable to be provided with a process thateliminates prion protein in specified risk materials from animal that islow in cost, is very stable, simple, easy to operate and which does notinterfere with regular farm operations.

SUMMARY

In accordance with the present disclosure there is now provided aprocess for degrading a prion protein in a specified risk materialcomprising the steps of feeding the specified risk material (SRM) to asequencing batch reactor (SBR) containing a layer of acclimatizedanaerobic sludge; and allowing the specified risk material to react withthe sludge at a temperature below 25° C. so as to allow degradation ofthe prion protein.

It is also provided a process of measuring the efficacy of a sequencingbatch reactor (SBR) to degrade prion proteins in a specified riskmaterial comprising the steps of feeding the specified risk material(SRM) to the sequencing batch reactor (SBR) containing a layer ofacclimatized anaerobic sludge; adding a model protein to the SBR; andallowing the specified risk materials and model protein to react withthe sludge at a temperature between 5° C. to 25° C., wherein degradationof the model protein is indicative of the efficiency of the SBR todegrade prion proteins in the SRM.

In a preferred embodiment, the specified risk material comprises animalcarcasses.

In another embodiment, the anaerobic sludge is derived from swinemanure, dairy manure and/or slaughterhouse sludge.

In a preferred embodiment, the specified risk material reacts with thesludge at a temperature between 5° C. to 25° C.; preferably at atemperature between 20° C. to 25° C.; more preferably at a temperatureof 20° C.

In another embodiment, the process of degrading a prion protein in aspecified risk material as described herein also comprises the step ofadding keratin to the SBR. The keratin can be from feather keratin orhoof keratin, and correspond to β-keratin or α-keratin. Preferably, thefeather keratin is from chicken feather and the hoof keratin is frombovine hoof.

In another embodiment, the SBR is an anaerobic digestion sequencingbatch reactor or a mesophilic anaerobic digestion sequencing batchreactor.

In another embodiment, the model protein described herein can be atleast one of perchloric acid-soluble protein, collagen, elastin andkeratin.

A “specified risk material” is intended to mean any material that maycontain prions concentrate therein, such as tissues of ruminant animals.Such material can include the brain, skull, eyes, trigeminal ganglia,spinal cord, vertebral column, dorsal root ganglia, and the tonsils anddistal ileum of the small intestine for example.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference will now be made to the accompanying drawings.

FIG. 1 illustrates a schematic representation of a laboratory scalesequencing batch reactor.

FIGS. 2A and B illustrate a graphic representation of feather keratindecrease in bags in reactors supplied with feather keratin.

FIG. 3 illustrates the gas production measured in reactors, and morespecifically in (A) the cumulative gas production and in (B) the dailygas production in reactors with (reactors 34 and 35) and without featherkeratin (reactors 13 and 14).

FIG. 4 illustrates in (A) the cumulative methane production, (B) aceticacid concentrations, (C) propionic acid concentrations and (D) propionicacid profiles measured in reactors with (reactors 34 and 35) and withoutfeather keratin (reactors 13 and 14).

FIG. 5A illustrates the isobutyric acid concentration measured inreactors with (reactors 34 and 35) and without feather keratin (reactors13 and 14).

FIG. 5B illustrates the soluble chemical oxygen demand (SCOD) measuredin reactors with (reactors 34 and 35) and without feather keratin(reactors 13 and 14).

FIG. 6 illustrates in (A) the pH profiles and in (B) the alkalinityprofiles measured in reactors with (reactors 34 and 35) and withoutfeather keratin (reactors 13 and 14).

FIG. 7 illustrates in (A) the total solids (TS), (B) volatile solids(VS) and (C) volatile suspended solids (VSS) profiles measured inreactors with (reactors 34 and 35) and without feather keratin (reactors13 and 14).

FIG. 8 illustrates in (A) the amount of keratin hydrolyzing organisms(KHOs) present in the mixed liquor of control reactor 13 and reactor 34with feather keratin and in (B) the correlation measured between thedegradation of feather keratin and the number of KHOs.

FIG. 9 illustrates in (A) the cumulative gas production and in (B) thedaily gas production measured in reactors with (reactors 15 and 16) andwithout feather keratin (reactors 13A and 13B).

FIG. 10 illustrates in (A) the cumulative methane production, (B) aceticacid concentrations, (C) propionic acid concentrations and (D)isobutyric acid concentrations measured in reactors with (reactors 15and 16) and without feather keratin (reactors 13A and 13B).

FIG. 11 illustrates the soluble chemical oxygen demand (SCOD) measuredin reactors with (reactors 15 and 16) and without feather keratin(reactors 13A and 13B).

FIG. 12 illustrates in (A) the pH profiles and in (B) the alkalinityprofiles measured in reactors with (reactors 15 and 16) and withoutfeather keratin (reactors 13A and 13B).

FIG. 13 illustrates in (A) the total solids (TS), (B) volatile solids(VS) and (C) volatile suspended solids (VSS) profiles measured inreactors with (reactors 15 and 16) and without feather keratin (reactors13A and 13B).

FIG. 14 illustrates the biodegradation of feather keratin in a PADseeded with swine manure (run 2).

FIG. 15 illustrates the biodegradation of hoof keratin in a PAD seededwith swine manure.

DETAILED DESCRIPTION

It is provided the use of psychrophilic anaerobic digestion sequencingbatch reactor (PADSBR) and mesophilic anaerobic digestion sequencingbatch reactor (MADSBR) technologies for eliminating prion in specifiedrisk materials (SRM), reducing the risk of biological contamination offauna, soil, groundwater and surface water.

Incineration is currently the primary method by which SRM are disposedin Europe. Laboratory results suggested that prions in SRM are unlikelyto survive incineration (Taylor et al., 1996, Neuropath Appl Neuro, 22:256-258). However, this treatment is expensive and precludes any valueto be derived from the SRM. Furthermore, incineration is impractical inmany regions due to the geographical dispersal of the cattlepopulations, which involves supplementary costs and unacceptablecontamination risks associated to the transportation ofprion-contaminated SRM.

Exceptional resistance of prions to dry heat has been observed inlyophilized samples or in samples dried onto glass or steel surfaces.Primary study revealed that gravity-displacement autoclaving at 134° C.for 1 h eliminates prions. However, paradoxical results were provided bystudies achieved with porous-load autoclaving: data suggested that thethermostability of prions was enhanced as the temperature of autoclavingwas increased (134 to 138° C.) (Taylor, 1999, Vet Microbiol, 67: 13-16).This indicates that increasing the temperature or exposure time ofprion-infected SRM to porous-load autoclaving would not be effective forreliable decontamination.

Ionizing, ultraviolet and microwave irradiations have little effect onprions (Taylor and Diprose, 1996, Neuropath Appl Neuro, 22: 256-258). Itis generally acknowledged that the inactivating effect of microwaves onmicroorganisms is due to the heat generated.

Strong sodium hypochlorite solutions or hot solutions of sodiumhydroxide seem to be the only methods that completely inactivate prions,whereas concentrated formic acid substantially reduces the infectivitylevel in histologically-fixed tissue. Prions are not completelyinactivated by exposure to autoclaving or sodium hydroxide solutions,but combining these two treatments can achieve inactivation. However,chemical treatments for inactivating prions are impractical forfull-scale, on-farm disposal strategies of bovine slaughterhouse sludgeor carcasses since the chemicals added to SRM may negatively affecttheir fertilizer value.

Concentrated formic acid solubilises proteins. As is the case forirradiations, there is little effect on prions after exposure toethylene oxide, glutaraldehyde, formalin, β-propiolactone oracetylethyleneimine.

The only detergent that has some effect on prions is sodium dodecylsulfate (SDS), but reports showed that SDS must be used only forcontaminated fluids (Taylor et al., 1999, Vet Microbiol, 67: 13-16).Therefore, treating SRM with SDS or other detergents seems impractical.

Organic solvents including ethanol, ether, acetone, 5% chloroform, 4%phenol, proprietary phenolic disinfectants and commercialsolvent-extraction processes involving hexane, heptane,perchloroethylene or petroleum are weakly effective for reducing prionsinfectivity.

Scrapie agents exposed to 3% hydrogen peroxide, chlorine dioxide, orperacetic acid were not or only slightly inactivated.

All these known treatment are associated with significant costs; in somecases protect prions from inactivation, hence enhancing theirthermostability; disrupt protein structure, rather than fixing it; andare inappropriate for the inactivation of prions in SRM due to potentialuser exposure at the farm and disposal problems onto agricultural land.

Consequently, biological treatments for the degradation of prions in SRMwhich are environmentally sound represent an alternative as describedherein.

Anaerobic digestion of organic matter is a process used for treatment oforganic waste and production of energy. In anaerobic bioreactors,organic matter is removed from the wastewater by the concerted action ofvarious groups of microorganisms. The activity of the microbial speciesparticipating in mineralization of organic matter is of crucialimportance for obtaining an efficient degradation of organic material inbioreactors. During anaerobic digestion, the organic matter is convertedvia acetate, short chained volatile fatty acids (like propionate,butyrate and iso-butyrate) and hydrogen/carbon dioxide to methane by theactivity of a complex microbial consortium consisting ofhydrolytic/fermentative acidogenic bacteria, acid-oxidizing bacteria andmethanogenic archaea. Four major groups of microorganisms have beenidentified with different functions in the overall degradation process.The first group represent the hydrolyzing and fermenting microorganismswhich are responsible for the initial attack on polymers and monomersfound in the waste material and produce mainly acetate and hydrogen, butalso varying amounts of volatile fatty acids (VFA) such as propionateand butyrate as well as some alcohols; group 2 represent thehydrogen-producing acetogenic bacteria which convert propionate andbutyrate into acetate and hydrogen; two groups (3 and 4) of methanogenicArchaea produce methane from acetate or hydrogen, respectively.

Bovine slaughterhouse sludge and carcasses are substrates composed ofreadily biodegradable organic matter that can be converted into a highquality biogas which can be processed into thermal and electricalenergy. Slaughterhouse wastes are well suited to anaerobic treatment andhigh solids removal is achieved. The effluent concentrations in heavymetal (cadmium, cobalt, nickel, copper, chromium) are below detectablelimit. Nutrients such as calcium, magnesium, sulphur and iron are inconcentration adequate for biological treatment of the wastewater.Slaughterhouse effluents are generally coagulated and flocculated toremove colloidal material and suspended solids responsible for the colorand turbidity (Al-Mutairi, 2006, Ecotoxicology and Environmental Safety,65: 74-83). Floc-forming compounds such as lime, alum, sodiumaluminates, ferric chloride and ferrous sulphate are used to removesuspended solids.

The sludge produced are mainly composed of blood, paunch, stomachcontents, undigested food, urine, loose meat, soluble proteins, fat,fecal matters, etc. The proportions of these elements in the sludge varybetween slaughterhouses. Al-Mutairi (2006, Ecotoxicology andEnvironmental Safety, 65: 74-83) indicated that alum and polymer used tocoagulate and flocculate solids from slaughterhouse effluents can betoxic to the microflora of a subsequent biological treatment process.When the concentrations of a coagulant exceed 100 mg/l in thesedimentation tank, the effluent is toxic to micro-organisms. Settledsludges are more contaminated and toxic than the sedimentation tanksupernatant.

Hygienic aspects are important when an external feedstock is brought ona livestock farm for co-digestion. Effluent from poultry and swineabattoir can be contaminated with biological contaminants. Pathogensremoval prior to land application is important to prevent reintroductionof contamination cycles in livestock production systems.

The fate of prions in anaerobic biological treatment has been reportedonly once (Kirchmayr et al., 2006, Wat Sci Technol, 53: 91-98). Noreduction of prion titer was observed in mesophilic conditions(Kirchmayr et al., 2006, Wat Sci Technol, 53: 91-98).

FIG. 1 is a schematic illustration of a laboratory scale sequencingbatch reactor (SBR) as used in the present disclosure and as describedin Canadian patent No. 2,138,091, the content of which is enclosedherewith by reference. Each of the reference numerals refer to thefollowing:

1. bioreactor;

2. sludge bed zone;

3. fill zone;

4. gas space;

5. biogas recirculation line;

6. biogas recirculation pump;

7. feeding line;

8. emptying port;

9. sludge sample port;

10. mixed liquor or supernatant sampling port;

11. gas outlet;

12. gas meter;

13. thermocouple; and

14. feeding system.

In the SBR shown in FIG. 1, the manure is loaded into the feeding system14 and fed to the bioreactor 1 through the feeding line 7. The manure isfed through the bottom of the bioreactor 1 which has been pre-inoculatedwith an anaerobic sludge 2. The biogas in the bioreactor 1 can berecirculated in the gas spaced 4 through the biogas recirculation line 5using the biogas recirculation pump 6.

It is thus described herein the use of SBR for decontaminating animalbrain, eliminating prions and reducing the risks of biologicalcontamination of soil, groundwater and surface water.

Experiments performed with the infectious isoform of prion proteins(PrPSc) require a biosafety level 3 laboratory. Proteins that resemblesPrPSc in their structure (number of amino acids, high proportion ofglycine residues, high content of β-sheet structures, high degree ofaggregation) and properties (protease- and heat-resistance, weaksolubility) have been selected to confirm the efficacy of SBR to degradeprion proteins in swine manure or dairy manure (Arai et al., 1996, JAppl Polym Sci, 60: 169-179; Caughey and Raymond, 1991, J Biol Chem,266: 18217-18223; Pan et al., 1993, Proc Natl Acad Sci USA, 90:10962-10966; Prusiner et al., 1998, Cell, 93: 337-348; Weissmann, 1999,J Biol Chem, 274: 3-6). Non-limiting examples of such suitable modelproteins to be added to an anaerobic digestion sequencing batch reactor(AD-SRB), in order to mimic and study the fate of prions in thesebioreactors, are disclosed hereinbelow.

Perchloric acid-soluble protein (PSP) has been isolated (14.149 kDaprotein with 137 amino acid residues) from rat kidney, brain and lung,as well as from rat, pig and chick livers (Hui et al., 2004, BiochemBiophys Res Commun, 321: 45-50). The secondary structure of that protein(2α-helices and 6β-sheets) resembles that of PrPSc (2α-helices and4β-sheets). PSP is heat-stable and proteinase K resistant, as is PrPSc.This suggests that PSP would be a suitable model protein for studyingthe fate of PrPSc in the AD-SBR.

Collagen is the most abundant protein in mammals, representing 25% oftotal proteins. Collagen is a major, fibrous, insoluble, extracellularprotein of skin, tendon, cartilage, bone and teeth, and serves to holdcells together.

Elastin is a fibrous cross-linked protein in the extracellular matrixthat provides elasticity for many tissues which can stretch to severaltimes their length and return to their original state when the tensionis released. Elastin is found in large amounts in the walls of bloodvessels and in ligaments, particularly in the neck of grazing animals.

Keratins forms one of the most diverse classes of fibrous proteins.There are various types of keratins, each composed of a complex mixtureof proteins. Keratins are major constituents of structures that growfrom the skin. α-Keratins are found in the hair (including wool), horns,nails, claws and hooves of mammals. A hoof is the tip of a toe of anungulate mammal, strengthened by a thick horny (keratin) covering. Thehoof consists of a hard or rubbery sole, and a hard wall formed by athick nail rolled around the tip of the toe.

α-Keratins are the most prevalent forms and are composed largely ofpolypeptide chains in the α-helix conformation. The harder β-keratinsare alternative forms that exist in the claws, scales and shells ofreptiles, and in the claws, scales, beaks and feathers of birds (Zubay,1988, Biochemistry, New York, N.Y., USA: Macmillan Publishing Company).β-Keratins are composed of stacked and folded β-sheets, and betterrepresent PrPSc. The amino acid sequences of β-keratins from variousbird species are highly similar and, like collagen and elastin, containa high proportion of glycine (Harrap and Woods, 1964, Biochem J, 92:19-26). Glycine-rich repeats are found in some, but not all, β-keratins.β-keratins are shorter (98 to 155 amino acids) than PrP proteins (256 to264 amino acids).

α-Keratins are composed of extremely long α-helical polypeptide chainsthat are arranged side by side to form fibers in a spiral fashion. Thepacking of α-helices is optimized and strengthened by wrapping ofindividual helices around each other. Many forms of α-keratins include(covalent) disulfide bonds formed between cysteine residues of adjacentpolypeptide chains, which confers permanent, thermally-stable crosslinking. The amount of disulfide bridges modulates strength and rigidityin keratins. Approximately one quarter to one third of feather keratinshave a β-conformation, mostly concentrated in the central, hydrophobicportion of the molecule. The rest of the protein forms an irregularmatrix. Closely related β-keratins are the main constituent of birdfeathers and account for about 90% of the feather rachis.

Slaughterhouse inoculums was used-herein, collected from asemi-industrial scale anaerobic sequence bench bioreactor (AD-SBR). TheSBR reactor is running at 20° C. and fed every week with fresh sludgefrom a commercial cattle slaughterhouse (Colbex, Levinoff, Québec) thatprocesses about 1000 cattle per day. Every week about 50 kg ofslaughterhouse sludge was collected and a representative sub-sample (1L) was stored at 20° C. for later analysis and what was left was used tofeed the semi-industrial SBR reactor.

The swine manure inoculums also used herein was collected from a semiindustrial scale anaerobic sequence bench bioreactor (AD-SBR).Similarly, it is kept at 20° C. and fed every week with fresh sludgefrom a commercial pig farm (1944 ch. Tremblay. Ste-Edwidge, Québec) thatprocesses about 4000 pigs per year. Every week about 50 kg swine manurewas collected and a representative sub-sample (1 L) was stored at 20° C.for later analysis and what was left was used to feed thesemi-industrial SBR.

It is also described the use of acclimatized anaerobic sludge asstarting material. When there is an important change in the bioreactor'sfeed composition, the bacterial sludge needs time to adapt to the newsubstrate. Sometime, the new substrate in the feed can inhibit thesludge microflora. Usually, the performance of the bacterial increaseswith time. The anaerobic sludge is acclimatized when the sludge reachesthe highest specific methane production (liter of methane per gram offeed) and when this maximal performance is maintained with time.

Feather is one of the large solid wastes in North America. Feathers aremainly composed of keratins. Keratins are insoluble and show highmechanical stability and resistance to proteolysis. Feather keratins area β-keratin composed of mainly stacked and folded β-sheets; therefore,feather keratins are poorly digested by common digestive enzymes, suchas trypsin and pepsin because of the high degree of cross-linking bydisulphide bonds, hydrogen bonding and hydrophobic interactions(Papadopoulos, 1986, Animal Feed Science and Technology, 14: 279-290). Asimilar structure is found in pathagenous protein (PrPsc). Therefore,chicken feathers are used as model proteins of PrPsc due to featherkeratins having a number of amino acids, high proportion of glycineresidues, high content of β-sheet structures, high degree of aggregationand properties (protease and heat resistance, weak solubility) similarto prion proteins.

Similarly, hooves are also used as model proteins of PrPsc due to theirhigh contant in hard keratins, insoluble and resistant to degradation bycommon proteolytic enzymes, such as trypsin, pepsin and papain becauseof their high degree of cross-linking by disulfide bonds, hydrogenbonding and hydrophobic interaction.

Degradation of feather keratin in AD-SBRs is demonstrated herein (seeTable 1 for exemplified conditions). Decrease in feather keratin in bagswas found in reactors as shown in FIG. 2A and FIG. 2B. Feather keratinin each reactor almost decreases linearly from 31 g at the beginning ofthe SBR run to 5 g within 120 days. On the contrary, no or very littledecrease in feather keratin was found in bags in reactors fed with watercontaining antibiotics. Addition of feather keratin affected the gasproduction in reactors. As shown in FIG. 3A, gas production in allreactors with and without feather keratin increased exponentially in thefirst 15 days. After 15 days, gas production in control reactorsgradually increased to the end of the experiment. No gas production wasobserved for a certain period. After the lag, gas production startedagain in reactors at a higher rate than in the control reactors. Inaverage, gas produced in reactors with feather keratin is 1.2 timeshigher than that produced in control reactors without feather keratin.Addition of feather keratin stimulated gas production.

The effect of feather keratin on gas productions could also be seen ondaily gas production rates. As shown in FIG. 3B, production rates in allreactors fluctuate within 3-23 L/day in the first 13 days. After that,gas production rates in control reactors gradually decreased to lessthan 1 L/day at the end of the experiment. However, gas production ratesin reactors with feather keratin still fluctuated from 0 to 7.7 L/dayafter 13 days and remain the same trend to the end of experiment. Theaverage rate measured in reactors is 1.6 times higher than that observedin control reactors.

Effect of feather keratin addition on gas production could also be seenon the cumulative methane production profiles (FIG. 4A). Methaneproduction in all reactors increased exponentially in the first 15-20days. After that, methane production in control reactors graduallydecreased to the end of the experiment. In average, reactors withfeather keratin produce 1.4 times more keratin than control reactors.The concentrations of volatile fatty acids (VFAs) including acetic acid,propionic acid and isobutyric acid in mixed liquor of all reactors werealso monitored during the SBR operation. The profiles of these VFAs wereshown in FIGS. 4B, C and D, correspondingly. In the first 10-15 daysconcentrations of all three organic acids are significantly higher. Thiscould be due the presence of fermentable organic substrates in startingsludge. After their consumption, the concentration of each WA fluctuatedwithin a certain range. Acetic acid fluctuated from 23 to 97 mg/L. Noobvious difference in acetic acid concentration could be observedbetween reactors with feather keratin and the control reactors in thefirst 60 day. The average acetic acid concentration in reactors withfeather keratin is 93 and 97 mg/L, respectively, higher than 75 and 68mg/L in control reactors. The result suggests that addition of featherkeratin stimulated production of acetic acid.

Propionic acid concentration was also affected by adding featherkeratin. As shown in FIG. 4C, propionic acid concentration in startingsludge is high. It gradually decreased in the first 15 days. After that,it fluctuated from 0 to 26 mg/L (see FIG. 4D). As observed in aceticacid profile, no significant difference in propionic acid productioncould be found between reactors with feather keratin and their controlreactors in the first 62 days. After 72 days values of propionic acidconcentration measured in reactors with feather keratin are mostlyhigher than those in control reactors. Addition of feather keratin didnot significantly affect isobutyric acid concentration (see FIG. 5A).After 90 days, most values of isobutyric acid concentrations measured inreactors with feather keratin are higher than in those in their controlreactors. The average concentration in reactors with feather keratin is1.9 time of that in control reactors, indicating a positive effect ofadding feather keratin.

Feather keratin also affects the concentration of soluble COD. As shownin FIG. 5B, SCOD in all reactors decreased gradually in the same trendin the first 60 days. Then, SCOD in control reactors kept decreasing tothe end of experiment. SCOD in reactors with feather keratin, however,increased gradually to the end of the experiment. The average SCODconcentration in reactors with feather keratin were of 8757 mg/L, whichis 1.1 times higher than that (8171 mg/L) measured in control reactors.

Feather keratin also affected mixed liquor pH in SBR reactors. As shownin FIG. 6A, pH in all reactors gradually decreased from 8.0-8.1 to 7.8in the first 50 days. After that, all the pH values measured in reactorswith feather keratin are 0.05-0.10 higher than those measured in controlreactors. Feather keratin significantly affected alkalinity. As shown inFIG. 6B, in the first 60 days alkalinity in all reactors mostlyfluctuated between 25000 and 27000 mg/L. After that, alkalinity measuredin control reactors still kept the same trend to the end of theexperiment. However, alkalinity in reactors with feather keratingradually increased up to 28839 and 28171 mg/L.

Feather keratin, in addition, also affected the TS profiles. As shown inFIG. 7A, TS in all control reactors gradually decreased in the first 80days then fluctuated within a certain range. Generally, TS in reactorswith feather keratin decreased at a faster speed than in controlreactors. TS values measured in reactors with feather keratin are mostlylower than those measured in control reactors. In average, TS value incontrol reactors is 1.1 times higher than in reactors with featherkeratin. Similar profiles observed in TS also exist in VS and VSSprofiles. VS and VSS in all reactors gradually decreased in the first60-80 days then fluctuated within a certain range. The significantdifference is that VS and VSS in reactors with feather keratin decreasedto a lower level than in control reactors. VS and VSS values measured inreactors with feather keratin are mostly lower than in reactors withfeather keratin.

Feather keratin in bags decreased in AD-SBRs fed with swine manure. Thedecrease observed is most due to microbial activity, i.e. keratinhydrolyzing microorganisms excreting keratinase and hydrolyzing thefeather keratin in bags. Keratin added in AD-SBRs fed with swine manuredid not affect negatively the performance of AD-SBRs. It improved thebiogas production and reduced biomass production of the ADSBRs.

BODIPY fluorescence exoenzyme staining methods were used to label andvisualize keratin hydrolytic microorganisms (KHOs). BODIPY FL casein wasfirst applied to samples of feather bags in bioreactors. After 30 mm ofstaining, fluorescence on bacteria with a morphotype of rod was observedon a weak fluorescent background. No other morphotype was observedduring 180 min staining. Sampling and microscopic examination werecarried out every 15 min. The background fluorescence, however,increased with time. The same staining was carried out on samples fromall the reactors with or without feather keratins added. When thesamples (fluids of feather bags or mixed liquor) from different AD-SBRswere heated at 100° C. for 10 min (as negative controls), nofluorescence (including background fluorescence) could be observed,indicating the fluorescence observed is due to microbiological activity.To specifically label the KHOs in AD-SBRs and not the consumers oflabelled hydrolysates which could be also stained in BODIPY FL staining,a set of inhibitors (Xia et al., 2008, FEMS Microbiology Ecology, 66:462-471) was added in all the staining incubations. Iodoacetate,fluoroacetate, and azide were added to inhibit the glycolysis, the TCAcycle, and the electron transport chain, respectively. The individualinhibitors were added at concentrations at which the energy metabolismsof all the microorganisms in AD-SBRs were effectively inhibited (Xia etal., 2008, supra). In the presence of the inhibitors, all fluorescingrods observed previously were still observed; the difference being thenumber of KHOs remarkably reduced after inhibition. Therefore, thebacteria positively stained are putative KHOs responsible for thedegradation of feather keratin observed in these AD-SBRs.

The relative numbers of KHOs in mixed liquor of AD-SBRs were estimatedby keeping all the procedure including sampling, sample preparation,microscopic examination, image capture and image analysis constant. Theresults are listed in Table 1. As shown in FIG. 8A, the numbers of KHOsin control reactor gradually decreased from 150 at day 0 to 56 at day113. The number of KHOs in reactor with feather keratin slightlydecreased in the first 20 days from 142 to 131, and then graduallyincreased up to 175 at day 85 before decreasing to 121 at day 113.

TABLE 1 Degradation of feather keratin and number of KHOs in AD-SBRs fedwith swine manure slurry Decrease percentage of feather Weight decreaseof feather keratin in bags (%) Number of Time keratin in bags (g)Reactor KHOs* (day) Reactor 34 Reactor 35 Reactor 34 35 Reactor (34) 031.76 ± 0.28 31.5 ± 0.44 0 0 0 22 25.17 ± 2.03   23 ± 0.44 21 27  12 ±38^(d) 55 16.12 ± 1.83 14.26 ± 0.01  51 55 49 ± 10 85  9.24 ± 0.11 8.37± 0.26 71 73 101 ± 37  113  4.54 ± 0.99 5.63 ± 0.23 86 82 81 ± 28

As the number of KHOs increased in the feather bags as well as theiractivity, a significant amount of soluble keratin was released into themixed liquor. Afterwards, KHOs in mixed liquor started to grow andmultiply and reached a high abundance (101) at the end of third monthand decreased after that, but still kept a relative high number (81) atthe end of four month because soluble keratin or its hydrolysates werekept released into the mixed liquor because once a bag was taken outfrom a SBR, a new feather bag was put in. The relative numbers of KHOsinside feather keratin bags of reactor were also counted. In order toeffectively harvest KHOs inside feather bags for BODIPI FL caseinstaining, numbers of bacterial cells obtained after centrifugation at800 g, 1000 g, 1500 g, 2000 g, 2500 g or 3000 g were estimated. Thebacteria obtained were stained with DAPI and their number was estimatedmicroscopically. The results are listed in Table 1. A positivecorrelation between the number of KHOs inside feather bags anddegradation of feather keratin was observed (see FIG. 8B). It confirmsthat degradation of feather keratin observed in bags was due to theactivity of KHOs visualized but not due to other physiochemicalreactions. The highest number of KHOs (101 cells) was observed in thethird month (day 85), corresponding to the highest degradation rate (71%in bio34 and 73% in bio35; Table 1) of feather keratin. Both the highestenrichment of KHOs and degradation rate were achieved in the thirdmonth. A slight decrease in the number of KHOs was observed at day 113.All results show that the hydrolysis rate is a limiting step for featherkeratin degradation in AD-SBRs fed with swine manure. These evidencesalso confirmed that the degradation of feather keratin was due toexo-enzyme secreted by KHOs rather than chemical and physical activitiesin AD-SBRs fed with swine manure.

BODIPY FL casein staining was also carried out on original sludgesamples from a commercial pig farm and a cattle slaughter house. Theresults are also shown in Table 2.

TABLE 2 KHOs in different AD-SBRs environments Chicken keratin SourcePresence Sample source Sample added stained of KHOs^(f) Commercial Rawswine NO Mixed − pig farm manure liquor Commercial cattle Raw slaughterNO Mixed − slaughterhouse farm house sludge liquor Lab-scale semiTreated swine NO Mixed + industrial bioreactor manure^(b) liquor 17Lab-scale semi Treated NO Mixed + industrial bioreactor slaughterhouseliquor 17a sludge^(c) Lab-scale bioreactor Inoculums of NO mixed + 13swine manure^(d) liquor Lab-scale bioreactor Inoculums of NO mixed + 14swine manure liquor Lab-scale bioreactor Inoculums of in bags insideof + 34 swine manure bags Lab-scale bioreactor Inoculums of in bagsmixed + 34 swine manure liquor Lab-scale bioreactor Inoculums of in bagsinside of + 35 swine manure bags Lab-scale bioreactor Inoculums of inbags mixed + 35 swine manure liquor Anaerobic bucket 1^(a) Water in bagsinside of − bags Anaerobic bucket 1 Water in bags Mixed − liquorLab-scale bioreactor Inoculums of NO mixed + 13A slaughterhouse liquorsludge^(e) Lab-scale bioreactor Inoculums of NO mixed + 13Bslaughterhouse liquor sludge Lab-scale bioreactor Inoculums of in mixedmixed + 15 slaughterhouse liquor liquor sludge Lab-scale bioreactorInoculums of in mixed mixed + 16 slaughterhouse liquor liquor sludge

No positive signal was observed. The KHOs observed in the AD-SBRs werenot present. BODIPY FL enzyme staining was also used to detect anypotential KHOs present in an anaerobic bucket in which feather bags wereincubated with distilled water under same condition as other AD-SBRs. NoKHOs were present in neither inside of bags nor mixed liquor (Table 2).

Feather keratin degradation in AD-SBRs is due to activity ofmicroorganisms. Microorganisms capable of hydrolyzing feather keratin(keratin hydrolyzing organisms, KHOs) are present in the swine manureused to feed the SBR reactors. When bags containing feather keratin areput into the reactors fed with swine manure, KHOs penetrate with liquidinto the bags and attach on the feather particles. Then, the KHOsexcrete extracellular keratinase and hydrolyze crystal feather keratininto soluble keratin, oligopeptides and amino acids. Once the aminoacids and/or oligopeptides are available, KHOs grow and multiply untiltheir number and activity reach a certain level, when amino acids andoligopeptides start to accumulate. The amino acids, oligopeptides andeven soluble keratin inside are driven outside of the bags by chemicalgradient, where they are used by microorganisms including hydrolyzers,fermenters, methanogenes and sulfate reducing bacteria. This processtake at least 30-40 days. During this period, therefore, no significancedifference in gas production, gas production rate, methane production,VFAs, pH, alkalinity, TS, VS and VSS is observed between the reactorswith feather keratin and the control reactors without feather keratin.

Once the extra amino acids and oligopeptides are obtained,microorganisms in mix liquor use them as nitrogen sources to grow and/orincrease activity. Hydrolysis and fermentation activities in thereactors are improved; therefore, a higher VFA (mainly acetic acid)concentration is detected in reactors with feather keratin. A high VFAlevel further stimulates the activity of methanogens. Methanogensconvert most of VFAs produced into methane and CO₂. Consequently, asobserved, more gas and methane, are produced in reactors with featherkeratin than in control reactors, which result in a low TS, VS and VSSvalues as a significant part of carbon source in the mixed liquor hasbeen transferred in gases. Because more CO₂ is produced in reactors withfeather keratin, a higher alkalinity level is detected in these reactorsthan in control reactors without feather keratin.

Addition of feather keratin affects the gas production. As shown in FIG.9A, cumulative gas production in reactors with feather keratin increasedexponentially in the first 40 days (up to ca 100 at day 40), while incontrol reactors gas production gradually increased to 32 L at day 40.In average, cumulative gas production in reactors with feather keratinis 6.7 times more than that produced in control reactors.

The effect of feather keratin on gas productions could also be seen ondaily gas production rates. As shown in FIG. 9B, gas production rates inall reactors fluctuate within 0-12 L/day in the first 60 days. Afterthat, gas production rates in control reactors gradually decrease toless than 1 L/day. The gas production rates in reactors with featherkeratin, however, still fluctuated within 0-2.5 L/day after the first 60days and remained the same trend to the end of experiment. The averagerate measured in reactors with feather keratin is 3.1 times higher thanthat observed in control reactors.

Effect of feather keratin addition on gas production could also be seenon the cumulative methane production profiles (FIG. 10A). Methaneproduction in reactors with feather keratin increased at much higherspeeds than that in control reactors. The average cumulative methaneproduction in reactors with feather keratin is 3.4 times more than thatproduced in control reactors. As shown in FIG. 10B, acetic acidconcentrations measured in all reactors with or without feather keratinfluctuates between 18-70 mg/L during all experimental period except inthe first 15 days where a much higher acetic acid concentration wasdetected in reactors with feather keratin. Similarly, no significantdifference in propionic acid as (FIG. 10C) well as isobutyric acidconcentration (FIG. 10D) could be detected between reactors with featherkeratin and control reactors. The concentrations of propionic acidvaried from 0 to 19 mg/L and the concentrations of isobutyric acidvaried between 0-16 mg/L. Therefore, feather keratin added does notsignificantly affect VFA production.

Addition of feather keratin did not significantly affect the solubleCOD. As shown in FIG. 11, SCOD in all reactors decreased gradually inthe same trend in the first 30 days before fluctuating from 1912 to 2185mg/L.

Similarly, addition of feather keratin did not significantly affectmixed liquor pH (FIG. 12A). Adding feather keratin, however, at leastslightly affected alkalinity. CaCO₃ measured in reactors with featherkeratin (FIG. 12B) are slightly higher than those measured in controlreactors. The average CaCO₃ concentration in reactors with featherkeratin is 15578 mg/L, higher than 13606 mg/L observed in controlreactors.

TS profiles in reactors with feather keratin are also different fromthose in control reactors without feather keratin. As shown in FIG. 13A,TS in the two control reactors gradually decreased from day 0 to day 20then fluctuated within 1.7-3.1%. However, TS in reactors with featherkeratin did not decrease and fluctuated between 2.8-7.9%. In average, TSvalue in reactors with feather keratin is 3.7%, being 1.6 times higherthan 2.3% measured in control reactors. The VS (FIG. 13B) and VSS (FIG.13C) profiles observed is similar to the TS profiles. VS and VSS valuesmeasured in reactors with feather keratin are all higher than thosemeasured in control reactors, in average being 1.6 (VS) and 1.7 (VSS)times, respectively, higher than those measured in control reactors.

Feather keratin degradation in the PAD seeded with swine manure andadded with feather bags (feather PAD) was measured to a level ofefficacy of 99% after 150 days (see Table 5, FIG. 14). It is disclosedthat keratinase is an inducible enzyme which can be induced in thepresence of keratin. Therefore a proper enrichment process increasesefficiency of the degradation of keratin in PADs.

Bovine hoof keratin degradation in the PAD seeded with swine manure andadded with hoof bags is also disclosed. In all individual nylon bags,86% of the hoof keratin was degraded after 113 days (see Table 6, FIG.15).

Therefore, it is demonstrated herein that feather keratin and bovinehoof can be hydrolyzed and degraded in AD-SBRs fed with at least swinemanure and slaughterhouse sludge. Addition of feather keratin improvesthe operation of AD-SBRs improving the biogas production. Prions incontaminated carcasses can thus be treated in AD-SBRs, since featherkeratin and/or bovine hoof and prions are similar in their structure.The enzymatic breakdown of prions would most importantly help revive theuse of animal meal as feed.

The present disclosure will be more readily understood by referring tothe following examples which are given to illustrate embodiments ratherthan to limit its scope.

EXAMPLE 1 Feather Preparation and Characterization

Freshly plucked white chicken feathers were collected from aslaughterhouse (rue Principale, Saint-Anselme, Québec) that processes900,000 chickens per week; and transferred to laboratory as soon aspossible. The chicken feathers were aliquot (2 Kg) into clean normalcotton bags and washed in a washing machine (delicate wash). The feathersamples were subsequently dried at 45° C. in a Unithern™ drier(Construction, CQLTD, England) until a constant weight was reached. Theywere cut and ground in a mill through a 4 mm screen. After that, thefeather samples were aliquot (around 33 g each) into nitrogen-freepolyester forage bags with a pore size of 50 microns (ANKOM Technology,2052 O'Neil Road Macedon, N.Y. 14502, USA), sealed with plastic tiewraps and washed again in washing machine using the same condition toget rid of as much dusts as possible. Finally, the washed feather bagswere dried again at 45° C. until their weights was constant. Featherbags were then attached into a steel stick and ready to be put into thebioreactors.

The feather samples ground were characterized physically and chemicallyin following ways. Total chemical oxidation demands (TCOD), ash contentand organic matter content were determined according to the standardmethods (APHA, 1992, In: Greenberg, A. E., Clesceri, L. S., Eaton, A. D.(Eds.), Standard Methods for the Examination of water and wastewater.American Public Health Association, Washington D.C.). Total Kjeldahlnitrogen (TKN) and ammonia-nitrogen were determined with a Tecator 1030Kjeltec auto-analyser (Tecator A B, Hoganas, Sweden) followingmacro-Kjeldahl method described standard methods. Protein concentrationwas calculated by multiplying the difference between TKN and ammonia-Nwith 6.25 (AOAC, 1984, In: Williams, S., Baker, D. (Eds.), OfficialMethods of Analysis of the Association of Official Analytical Chemists,Arlington, Va.). Fat content was determined according to Schrooyen etal. (Schrooyen et al., 2000, Journal of Agricultural and Food Chemistry,48: 4326-4334). Chicken feather samples (30 g) were Soxhlet extractedfor approximately 12 h with petroleum ether (boiling range 40-60° C.)and the fat extracted was measured (Schrooyen et al., 2000, supra).

Amino acid concentration was determined following the isotope dilutionmethod described by Calder et al. (Calder et al., 1999, RapidCommunications in Mass Spectrometry, 13: 2080-2083). Briefly, rawfeather samples were hydrolysed with 50 ml of 6 mol/L phenol-HCl at 110°C. for 24 h and the hydrolysate was filtrated. Then, 2 g hydrolysate wasdiluted with 3 g of ultra-pure water; 1 g of this solution was combinedwith a mixture (200 mg) of labeled amino acids (¹³C and ¹⁵N amino acidsisotope standards, CDN Isotopes, Pointe-Claire, Que., Canada; CambridgeIsotope Laboratories Inc., Andover, Mass., USA) which serves as aninternal standard. Samples of the deproteinised plasma and of thehydrolysate were eluted through a poly-prep chromatography column (Resin100-200 mesh H, BIO RAD, Hercules, Calif., USA) and derivatised withN-(tertbutyldimethylsilyl)-Nmethyltrifluoroacetamide (MTBSTFA) anddimethylformamide (DMF) (Sigma-Aldrich, Ontario, Canada) in a 1:1 ratio.Measurements of [2-15N] Lys and of the amino acids in processed sampleswere performed using gas chromatography-mass spectrometry (GC-MS, ModelCG6890-MS5973, Hewlett Packard Co., Wilmington, Del., USA).

EXAMPLE 2 SBR Setups

Eight 42 L Plexiglas SBRs were used. FIG. 1 is a schematicrepresentation of these digesters. Once loaded, the SBRs consist of asolid, liquid and gas phase. Individual parts are set up on each phaseof the SBRs to take samples. The starting sludge volume at the beginningof each cycle was 35 L. All SBRs were operated in a room kept at 25° C.The sludge load for both swine manure and slaughterhouse sludge is 1.5 gCOD/L-d. The loading rate of feather keratin was determined according tothe limited space of cylinder of AD-SBRs and the total oxygen demand(TCOD) concentration of feathers. The loading rate of feather keratin(adding in bags or adding in mixed liquor) in all SBRs was kept as same(0.21 g COD/L.d). To load sludge into the SBRs, 160 L swine manure orslaughterhouse sludge, was withdrawn from the semi-industrial scalereactors and transferred into a 200 L barrel. A paint mixer was used tokeep solids suspended during sludge aliquot. A 5 L container was used toaliquot sludge. Finally, 35 L sludge was transferred into each of the 8SBRs (4 fed with swine manure and the other 4 with slaughterhousesludge, respectively.) Before sampling for physiochemical analysis, thereactors were mixed by circulating the biogas for 5-10 min beforesamples were withdrawn.

All SBR runs and their running conditions are shown in Table 3. Two SBRruns have been carried out. In Run 1, 4 SBR reactors were used toexamine degradation of chicken feather in AD-SBRs fed with swine manure.In Run 2, another 4 SBR reactors were used to investigate degradation ofchicken feather in AD-SBRs fed with slaughterhouse sludge. Chickenfeathers were added in two ways. As shown in Table 3, feather sampleswere kept in bags in Run 1 and directly mixed with slaughterhouse sludgein Run 2. Feather bags were incubated with distill water in a sealedbucket (6 bags in each) at 25° C. as a negative control.

TABLE 3 AD-SBR running conditions Feather SBR Feather protein SBRreactor adding load run label Specification Sludge source mode (gCOD/L-d) 1 13 Control 1 Swine manure In bags 0 (−feather) 14 Control 2Swine manure In bags 0 (−feather) 34 Positive 1 Swine manure In bags0.21 (in (+feather) 12 bags) 35 Positive 2 Swine manure In bags 0.21 (in(+feather) 12 bags) 2 13A Control 1 Slaughterhouse Mixed in 0 (−feather)sludge sludge 13B Control 2 Slaughterhouse Mixed in 0 (−feather) sludgesludge 15 Positive 1 Slaughterhouse Mixed in 0.21 (+feather) sludgesludge 16 Positive 2 Slaughterhouse Mixed in 0.21 (+feather) sludgesludge

EXAMPLE 3 Physiochemical and Microbiological Characterization

All physiochemical factors measured for the AD-SBRs and their samplingfrequencies are listed in Table 4.

TABLE 4 Physiochemical factors monitored Factors analyzed Samplingfrequency In mixed liquor: Once a week TS (total solids), VS (volatilesolids), VSS (Monday) and end (volatile suspended solids), nitrogenincluding of the treatment NH₃—N and TKN (total nitrogen), pH &alkalinity, cycle (Thursday) TCOD (total chemical oxygen demand), SCOD(soluble chemical oxygen demand), VFA (volatile fatty acids). In bagscontain chicken feather: Once at the Weight, TCOD, TKN, NH₃—N, TS, VS,FS, beginning Once amino acids a month in further treatment cycle(weight) Biogas: Total volume once a Total volume, composition (CH₄,CO₂, H₂S) day Composition once a Week

The pH, alkalinity, total solids (TS), volatile solids (VS) and volatilesuspended solids (VSS) were determined according to standard methods(APHA, 1992, supra). The value of pH was measured using a pH meter.Alkalinity was measured with titration to pH 4.38. TS content wasdetermined by drying a 10 ml sub-sample for 24 h at 105° C. Dried solidswere incinerated for 3 h at 550° C. for VS measurement. Similarly,centrifuged slurry was used to determine VSS through incineration for 3h at 550° C. Soluble chemical oxygen demand (SCOD) was determined byanalyzing the supernatant of centrifuged slurry. Total chemical oxygendemand (TCOD) and soluble chemical oxygen demand (SCOD) were determinedusing the closed reflux colorimetric method (APHA, 1992, supra). Featherbags were taken off and weighed in each month. A new feather bag of thesame weight was put in a SBR when a feather bag was taken out. Biogasproduction was monitored daily using wet tip gas meters and itscomposition (methane, carbon dioxide, hydrogen sulfide, and nitrogen)was analyzed weekly using a Hach Carle 400 AGC gas chromatograph (Hach,Love-land, Colo.). The column and thermal conductivity detector wereoperated at 80° C. Total nitrogen (TKN) and ammonia-N were determinedusing an auto-analyzer according to the macro-Kjeldahl method (APHA,1992, supra) with a Tecator 1030 Kjeltec auto-analyzer (Tecator A B,Hoganas, Sweden). Volatile fatty acids (VFA) including acetic,propionic, butyric, isobutyric, isovaleric, valeric and caproic acids)were analyzed using an AutoSystem™ gas chromatography equipped with ahigh resolution megabore column (Perkin-Elmer Corporation; Norwalk,Conn. 06859, USA) connected to a flame ionization detector (Masse etal., 2000, Bioresource Technology, 75: 205-211; Masse et al., 2008,Biorescource Technology, 99: 7307-7311).

EXAMPLE 4

Chicken Feather Keratin (β-Keratin) Biodegradation in PsychrophilicAnaerobic Digestion Sequencing Batch Reactors (PADs) Inoculated withSwine Manure Sludge

Freshly plucked white chicken feathers were collected from aslaughterhouse (Principale Street, Saint-Anselme, QC, Canada) andtransferred to the laboratory within 4 hours. The chicken feathers weredivided into aliquot (2 kg each) parts in clean cotton bags and washed(delicate cycle) in a washing machine (Frigidaire, Martinez, Ga., USA)with tap water. The feather samples were then dried at 45° C. in aUnithern dryer (Construction CQLTD, England) until a constant weight wasreached (about eight weeks). The samples were ground in a mill(Thomas-Wiley Laboratory Mill), screened through a 4-mm screen and thendivided into aliquot (around 33 g each) parts in nitrogen-free polyesterforage bags with a pore size of 50 μm (ANKOM Technology, Macedon, N.Y.,USA). The bags were sealed with plastic tie wraps and washed in thewashing machine again to remove as much dust as possible. Finally, thewashed feather bags were dried at 45° C. until their weights remainedconstant. When needed, 12 dried feather bags (each containing around 31g ground feathers) were attached onto a steel stick and inserted into aPAD.

Three 42-L Plexiglas PADs were used in this study. Two of the PADs wereinoculated with anaerobic sludge adapted to swine manure. The inocula ofthe PADs, representing 100% of the volume, came directly from a 7-m³semi-industrial anaerobic bioreactor that was located at Agriculture andAgri-Food Canada's Dairy and Swine Research and Development Centre(Lennoxville, QC, Canada) and had been treating swine manure at 25° C.over a 2-year period. Twelve feather bags (starting load 4 g/g VSsludge) were added in one of them and the other without adding featherbags was used as a negative control. The third PAD was filled withdeionized water containing antibiotics (ampicillin at a finalconcentration of 100 μg/mL) and added with 12 feather bags to determinethe physiochemical loss of feathers from the feather bags. The volume ofthe inoculum and deionized water for each PAD was 35 L. All PADs wereoperated in a room kept at 25° C. In run 1, every 30 days, three featherbags were taken out of each PAD, washed with deionized water, dried for48 h at 45° C., and weighed. Run 2 was start when run 1 was processingin the third month, 12 new feather bags were added into bioreactor andfollowing the same experimental procedure as run 1, the weight deductionof run 2 was also recorded.

TABLE 5 Biodegradation of feather keratin in a PAD seeded with swinemanure sludge in run 2 Weight Day(s) of feathers (g) Weight lost (%) 03.1.4 ± 0.1    0 22 NA NA 55 NA NA 85   2 ± 0.6 94 113 0.4 ± 0.2 98 1500.14 ± 0.08 99

Feather keratin degradation in the PAD seeded with swine manure andadded with feather bags (feather PAD) was recorded. In Run 1, 86% (from31±0.3 g at the beginning of the experiment to 5±1.6 g at the end of theexperiment) of the feather keratin was degraded after 113 days. Incontrast, only a small reduction (<3%) of the feather keratin wasobserved in the PAD containing water with antibiotics (data not shown).In Run 2, 99% (from 31±0.1 g at the beginning of the experiment to0.14±0.08 g at the end of the experiment) of the feather keratin wasdegraded after 150 days (Table 5; FIG. 14). The feather degradation ratein run 2 is higher than in Run 1, indicating that keratin degradingorganisms (KDOs) were successfully enriched in Run 1. All result foundin this study also demonstrated that the keratinase was an inducibleenzyme which can be induced in the presence of keratin. Therefore aproper enrichment process is preferable for efficient degradation ofkeratin in PADs.

EXAMPLE 5 Bovine Biodegradation in Psychrophilic Anaerobic DigestionSequencing Batch Reactors (PADs) Bovine Hooves Preparation

Bovine hooves (α-keratin) were collected from a local slaughterhouse(Colbex, Levinoff, Quebec) that processes about 1000 cattle per day andtransferred to the laboratory within 4 hours. The bovine hooves werethen washed with deionized water and dried at 45° C. in a Unithern dryer(Construction CQLTD, England) until a constant weight was reached (aboutone week). Subsequently, the bovine hooves were manually cut into piecesof 3-5 cm in diameter with a drill and ground in a mill (Thosmas-Wiley,Laboratory Mill) through a 2-mm screen. The samples obtained werefurther ground in a blender (Vita-Mix5200, Vitamix Corporation) and wentthrough a sieve with a pore size of 500 μm. After that, 27 g homogenizedbovine hooves were aliquot into nitrogen-free polyester forage bags witha pore size of 50 microns (ANKOM Technology, 2052 O'Neil Road Macedon,N.Y. 14502, USA), sealed with plastic tie wraps; and washed in a washingmachine (Frigidaire, Martinez, Ga., USA) using delicate cycle to removeas much dusts as possible. The washed hoof bags were dried again at 45°C. until their weights kept constant. Finally, 12 hoof bags (ca: 27 geach) were attached into a steel stick and put into the bioreactors.

PAD Set Up

Three 42-L Plexiglas PADs were used in this study. Two PADs wereinoculated with anaerobic sludge adapted to swine manure. The inocula ofthe PADs, representing 100% of the volume, came directly from a 7-m³semi-industrial anaerobic bioreactor that was located at Agriculture andAgri-Food Canada's Dairy and Swine Research and Development Centre(Lennoxville, QC, Canada) and had been treating swine manure at 25° C.over a 2-year period. Twelve hoof bags were added in one of them and theother without adding hoof bags was used as a negative control. The thirdPAD was filled with deionized water containing antibiotics (ampicillinat a final concentration of 100 μg/mL) and added with 12 hoof bags todetermine the physiochemical loss of bovine hooves from the hoof bags.The volume of the inoculum for each PAD was 35 L. All PADs were operatedin a room kept at 25° C. Every 30 days, three hoof bags were taken outof each PAD, washed with deionized water, dried for 48 h at 45° C., andweighed. The weight deduction was recorded.

Bovine hoof keratin degradation in the PAD seeded with swine manure andadded with hoof bags was recorded. In all individual nylon bags, 86%(from 27.6±0.1 g at the beginning of the experiment to 3.9±0.2 g at theend of the experiment) of the hoof keratin was degraded after 113 days(Table 6; FIG. 15). In contrast, only a small reduction (<10%) of thehoof keratin was observed in the PAD containing water with antibiotics.

TABLE 6 Biodegradation of bovine hoof keratin in a PAD seeded with swinemanure Day(s) Weight of bovine hooves (g) Weight lost (%) 0 27.6 ± 0.1  0 22 17.3 ± 1.4  37 55 NA NA 85 7.7 ± 0.6 72 113 3.9 ± 0.2 86

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A process for degrading a prion protein in a specified risk materialcomprising the steps of: (a) feeding the specified risk material (SRM)to a sequencing batch reactor (SBR) containing a layer of acclimatizedanaerobic sludge; and (b) allowing the specified risk material to reactwith the sludge at a temperature below 25° C. so as to allow degradationof the prion protein.
 2. The process of claim 1, wherein the specifiedrisk material comprises animal carcasses.
 3. The process of claim 1,wherein the anaerobic sludge is derived from swine manure or dairymanure.
 4. The process of claim 3, wherein the dairy manure is derivedfrom slaughterhouse sludge.
 5. The process of claim 1, wherein thespecified risk material reacts with the sludge at a temperature between5° C. to 25° C.
 6. The process of claim 5, wherein the specified riskmaterial reacts with the sludge at a temperature between 20° C. to 25°C.
 7. The process of claim 5, wherein the specified risk material reactswith the sludge at a temperature of 20° C.
 8. The process of claim 1,further comprising the step of adding keratin to the SBR.
 9. The processof claim 8, wherein the keratin is from feather keratin or hoof keratin.10. The process of claim 9, wherein the keratin is β-keratin orα-keratin.
 11. The process of claim 9, wherein the feather keratin isfrom chicken feather.
 12. The process of claim 9, wherein the hoofkeratin is from bovine hoof.
 13. The process of claim 1, wherein the SBRis an anaerobic digestion sequencing batch reactor.
 14. The process ofclaim 1, wherein the SBR is a mesophilic anaerobic digestion sequencingbatch reactor.
 15. A process of measuring the efficacy of a sequencingbatch reactor (SBR) to degrade prion proteins in a specified riskmaterial comprising the steps of: (a) feeding the specified riskmaterial (SRM) to the sequencing batch reactor (SBR) containing a layerof acclimatized anaerobic sludge; (b) adding a model protein to the SBR;and (b) allowing the specified risk materials and model protein to reactwith the sludge at a temperature between 5° C. to 25° C., whereindegradation of the model protein is indicative of the efficiency of theSBR to degrade prion proteins in the SRM.
 16. The process of claim 15,wherein the model protein is at least one of perchloric acid-solubleprotein, collagen, elastin and keratin.
 17. The process of claim 16,wherein the keratin is β-keratin or α-keratin.
 18. The process of claim16, wherein the keratin is from feather keratin or hoof keratin.
 19. Theprocess of claim 18, wherein the feather keratin is from chickenfeather.
 20. The process of claim 18, wherein the hoof keratin is frombovine hoof.